Quantifying bacterial efflux within subcellular domains of Pseudomonas aeruginosa

Molecular efflux is a mechanism through which bacteria actively expel undesirable substances. This is a crucial line of defense against toxic chemicals in harsh environments. Understanding how efflux works is critical for designing antimicrobial strategies. Though much is already known about efflux proteins, important details about the mechanisms of efflux (e.g., importance of specific subcellular domains and ejection rates) have yet to be experimentally quantified. Herein, we use the nonlinear optical technique, second harmonic light scattering, to simultaneously measure the efflux rates from the periplasm and cytosol of a Gram-negative bacterium. The influence of efflux on the uptake kinetics of a mild antimicrobial, malachite green (MG), by Pseudomonas aeruginosa was quantified. It is observed that efflux primarily occurs from the periplasm and is two orders of magnitude faster than from the cytosol. Efflux pumps activate to maintain MG concentrations in the periplasm below 1 µM, while efflux from the cytosol maintains MG concentration below 0.1 µM. Efflux pumps are shown to saturate when exogenous MG concentrations are greater than 25 µM, while the cytosol efflux function saturates at >15 µM. Finally, efflux pumps can simultaneously eject different compounds, as proven by experiments with both MG and hexane, a known effluxable compound.IMPORTANCEMolecular efflux pumps are a crucial defense mechanism that protects bacteria from an otherwise unchecked influx of toxic molecules present in the extracellular environment. The efflux functions constitute a significant hindrance to antimicrobial efficacy. While much is now known regarding the structure of these channels, knowledge of the influence of efflux in individual subcellular domains and the associated ejection rates is still lacking. Using the nonlinear optical technique, second-harmonic light scattering, we have measured the threshold concentrations for pump activation, saturation concentrations, and efflux rates from both the periplasm and cytosol in living Gram-negative bacteria. The quantified efflux data in the different subcellular compartments not only provide a clear mechanistic understanding but also are critical for developing antimicrobial strategies.